A vascularized three-dimensional model integrating primary breast tumor cells and microvascular fragments: mimicking the tumor microenvironment involved in chemoresistance

BackgroundTumorigenesis is a complex and dynamic process in which the tumor microenvironment (TME) plays a central role. In solid tumors, the TME contributes to key mechanisms of tumor progression, including metastasis, immune evasion, and resistance to therapies. One major challenge in preclinical cancer research is the development of reliable three-dimensional (3D) in vitro models, which more accurately replicate the in vivo tumor architecture and microenvironmental conditions, such as hypoxia and extracellular matrix (ECM) organization. However, reproducing functional vascular networks and neo-angiogenesis within these models remains a key challenge.MethodsIn this study, an advanced 3D tumor model, referred to as angiotumoroids, was developed by co-culturing primary murine breast tumor cells (PTCs) with species-specific adipose-derived microvascular fragments (MVFs). Angiotumoroids were characterized using scanning electron microscopy and immunostaining, and angiogenesis was evaluated through collagen gel sprouting assays. High-resolution proteomic profiling was conducted, focusing on signatures associated with angiogenesis, extracellular matrix (ECM) composition, and tissue remodeling. Additionally, the response and internalization to anticancer drug treatments were evaluated.ResultsMVFs are successfully integrated in angiotumoroids, resulting in the formation of vasculature-like structures and demonstrating robust structural organization with dynamic modulation of matrix metalloproteinase 9. Formation of neovasculature was visualized through sprouting and branching, driven by both direct PTC-MVF interactions and PTC-conditioned media, highlighting the roles of juxtacrine and paracrine signaling. Proteomic profiling revealed distinct expression patterns associated with angiogenesis, ECM components (including collagen types I and IV), and active ECM remodeling with elevated MMP expression. Additionally, angiotumoroids showed increased expression of ATP-binding cassette (ABC) transporters, particularly ABCB1 (P-glycoprotein), suggesting potential mechanisms of drug efflux. Functionally, angiotumoroids demonstrated reduced sensitivity to doxorubicin compared to PTC spheroids, maintaining structural integrity and higher cell viability post-treatment. Time-course analysis revealed preferential doxorubicin accumulation in MVF-enriched regions, as confirmed by colocalization with CD31, indicating a spatially regulated distribution of the drug mediated by the vascular compartment.ConclusionsCollectively, these findings establish angiotumoroids as a robust and physiologically relevant in vitro model for studying tumor vascularization, ECM dynamics, and therapeutic response. This platform holds significant promise for predictive cancer research and preclinical drug screening, bridging the gap between traditional in vitro systems and in vivo models.